This disclosure relates generally to the field of immunoassays and specifically to immunoassays used to determine concentrations of thyroxine (T.sub.4), triiodothyronine (T.sub.3) and thyroxine-binding globulin (TBG) in fluid samples such as blood serum samples.
It is presently thought that the concentration of free T.sub.4 (FT.sub.4) in a blood serum sample may be more significant clinically than the concentration of total T.sub.4 which includes both free (or unbound) T.sub.4 and T.sub.4 which is bound to serum proteins, especially TBG. Free T.sub.3 concentration may also have clinical significance. Since about 99.97% of the T.sub.4 (and 99.7% of T.sub.3) in normal human serum consists of T.sub.4 (or T.sub.3) that is bound to serum proteins, one indirect method of measuring either free thyroid hormone (unbound) involves measuring for total thyroid hormone by known means (e.g. immunoassays) and then, by using known binding constants, simply calculating the amount of free T.sub.4 (or T.sub.3) present. Unfortunately, the use of binding constants to indirectly determine free T.sub.4 (or free T.sub.3) is not always useful in determining the free thyroid hormones in abnormal human serum. Hence, present methods for determining free T.sub.4 or free T.sub.3, especially abnormal amounts, are commonly based on an equilibrium dialysis which is a very time consuming method. Quite surprisingly, we have found that either free thyroid hormone can now be rapidly measured by slightly modifying present immunoassay methods for detecting total T.sub.4 or total T.sub.3. Details of our method are described herein.